Berangan Effect of benzylaminopurine (BAP) pulsing on in vitro shoot multiplication of Musa acuminata (banana) cv.
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African Journal of Biotechnology Vol. 10(13), pp. 2446-2450, 28 March, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1149
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Effect of benzylaminopurine (BAP) pulsing on in vitro
shoot multiplication of Musa acuminata (banana) cv.
Berangan
Najmeh Jafari, Rofina Yasmin Othman and Norzulaani Khalid*
Centre for Biotechnology in Agriculture Research/Institute of Biological Sciences, Biotechnology and Bioproduct
Research Cluster, Faculty of Science, University Malaya, 50603 Kuala Lumpur, Malaysia.
Accepted 12 November, 2010
MS (Murashige and Skoog) media supplemented with benzylaminopurine (BAP) showed that the
number of bud formation in shoot cultures of Musa acuminata cv.Berangan during the initiation stage
increased proportionately with the concentrations used (11, 22 and 33 µM). However, the highest
concentration of BAP (33 µM) simultaneously increased the formation of abnormal shoots. After the
first apical bud appeared, explants were transferred to MS medium with lower concentrations of BAP
either with or without indole acetic acid (IAA). Proliferation media supplemented with IAA showed
enhanced shoot multiplication and elongation but did not help to reduce the abnormality index that
occurred.
Key words: micropropagation, adventitious buds, banana, Musa acuminata
INTRODUCTION
Banana originated from the South East Asian region, banana through in vitro techniques has been reported by
where the greatest diversity of edible bananas are found several workers using different explants sources and
(Stover and Simmonds, 1987). Bananas account for methods (Jalil et al., 2003; Madhulatha et al., 2004;
approximately 22% of the fresh fruit production and are Strosse et al., 2006; Wong et al., 2006; Venkatachalam et
ranked as the second most important fruit crop (Pua, 2007). al., 2006, 2007; Resmi and Nair, 2007; Shirani et al., 2009).
The different types of banana have not been fully In tissue culture, plant growth regulators (PGR) are
exploited since Cavendish types mainly dominate the critical media components in determining the develop-
market place globally. mental pathway of the plant cells. Cytokinins such as
For commercialization, it is important that consistent benzylaminopurine (BAP) and kinetin are generally
supplies of good quality bananas are produced. This could known to reduce the apical meristem dominance and
be achieved through clonal planting materials obtained induce both axillary and adventitious shoots formation from
through tissue culture propagation technique. This tech- meristematic explants in banana (Madhulatha et al.,
nique provides high rates of multiplying genetically uniform, 2004). The most established banana shoot-tip culture
pest and disease-free planting materials. Propagation of system was achieved by using BAP as a supplement to
Murashige and Skoog (MS) basal media (Murashige and
Skoog, 1969). The effectiveness of BAP over other cyto-
kinins in inducing multiplication of shoot tip cultures has
*Corresponding author: E-mail: lani@um.edu.my. Tel: +603- been reported in different cultivars of bananas (Rahman
79677806. Fax: +603-7967. et al., 2006; Resmi and Nair, 2007; Farahani et al., 2008;
Buah et al., 2010). BAP has a marked effect in stimula-
Abbreviations: PGR, Plant growth regulators; BAP, ting the growth of axillary and adventitious buds and foliar
benzylaminopurine; IAA, indole acetic acid; MS, Murashige and development of shoot tip cultures (Abeyarante and Lathiff,
Skoog; IBA, indole-3-butyric acid. 2002; Buah et al., 2010). Meanwhile, combinations ofJafari et al. 2447
Table 1. Effect of different concentration of BAP on proliferation Proliferation media
of adventitious buds.
The explants were subsequently transferred to MS medium with
Concentration of Fresh weight Bud formation either the same or lower concentrations of BAP than the initiation
media and supplemented either with or without IAA (Table 2).
BAP (µM) (g) (%)
Transfers to fresh medium and subculturing were carried out every
11 0.88± 0.25 a 45 30 days up to 3 cycles. In the propagation stage, the shoots per
22 2.54± 0.36 b 75 explant were counted and the average shoot length from randomly
selected sample was determined.
33 4.04± 0.45 c 90
Abnormality index was calculated by taking the ratio of abnormal
Values are the mean ± SD. Values with different letters are to normal shoots. Plantlets were categorized as normal or abnormal
significantly different at the 0.05 level. based on morphological appearance such as hyperhydricity and
presence of undifferentiated tissue. Data were analyzed using
SPSS 14.0 version and the mean difference were contrasted using
Duncan’s multiple range tests at the 0.05 level of significance.
BAP with auxins such as indole acetic acid (IAA) or
indole-3-butyric acid (IBA) were also used for in vitro
multiplication of bananas (Dhed’a et al., 1991; Resmi and Culture conditions
Nair, 2007). Although BAP stimulates shoot proliferation
The pH of all culture media was adjusted to 5.8 prior to addition of
in bananas, it is also known to have mutagenic effects at gelrite at 2.5 g/l and autoclaving. All culture media were autoclaved
high concentration producing off type plantlets (Bairu et at 121°C for 20 min. Cultures were maintained at 28°C under 16-h
al., 2008). Appearance of the off-type plantlets from in photoperiod with light intensity of 31.4 µmol m-2 s-1.
vitro multiplication process is considered a great dis-
advantage. The purpose of this study is to develop a
regime for the use of BAP that is widely used to increase RESULTS AND DISCUSSION
the multiplication rate for plantlets and concurrently
control its mutagenic effect so as to decrease the percen- The effects of different concentration regimes of BAP on
tage of morphologically abnormal plantlets formation. bud initiation and shoot multiplication either with or without
IAA were investigated. After the initial bud was sub-
cultured, multiple adventitious buds were produced from
MATERIALS AND METHODS the base of the explants after 30 days. Table 1 shows the
fresh weight and percentage of buds obtained on media
Plant materials
tested. The frequency of bud formation doubled and the
Young suckers of Musa var. ‘Berangan’ were collected from a fresh weight increased about four times higher in media
healthy true-to-type mother plants. After removing the leaves and with BAP at 33 µM when compared to media supplemen-
the roots, the suckers were thoroughly washed with tap water to ted with 11 µM BAP.
remove adhering soil. The samples were trimmed to a size of 8 to Explants with newly formed buds were transferred to
10 cm in length and 5 to 6 cm in diameter by removing the layers of media with either similar or lower concentrations of BAP
the developing leaves. Then, the suckers were placed under
running tap water for 30 min before the shoot apices were transferred at the proliferation stage. Results indicated that BAP at
to a laminar flow. The shoot apices explants were sequentially 22 µM induced the highest number of normal and
treated with 50% (v/v) Chlorox (the active ingredient is 5.25% elongated shoots, although more shoots proliferated on
sodium hypochlorite) mixed with few drops of Tween 20 for 30 min initiation medium supplemented with BAP at 33 µM (Table
in order to sterilize the surface. Then they were dipped into 95% 2). At 11 and 6.5 µM BAP, the lowest number of
(v/v) ethanol for 2 min followed by a mixture of 100% (v/v) Chlorox,
Tween 20 and 0.1% (w/v) HgCl2 solution for 5 min. After these
elongated shoots was obtained. With the addition of IAA
treatments, explants were rinsed with sterilized distilled water and to BAP supplemented media, shoot proliferation and
left to dry for 15 min. Subsequently, the explants were trimmed to a elongation were generally enhanced with an optimal con-
size of 3 to 4 cm in length and 2 to 3 cm in diameter. centration of 22 µM BAP and 2 µM IAA where the number
of shoots was 20.25 ± 2.5 and shoot length was 10.60 ± 1
cm.
Initiation media
The abnormality index of proliferated shoots varied with
different concentrations of BAP either with or without IAA
The culture initiation media was prepared by adding 10 mg/l ascorbic
acid (as an antioxidant), 87 µM sucrose and 2 g /l gelrite as (Figure 1). The results indicated that with decreasing BAP
solidifying agent to MS basal medium (Murashige and Skoog 1962) concentration in the proliferation stage, abnormality index
and supplemented with different concentrations of BAP (11, 22 and significantly decreased. There was no significant difference
33 µM). Cultures were maintained on each BAP concentration until in abnormality index between BAP and combination of
the first individual bud appeared. Subsequently, each explant was BAP with IAA. The highest abnormality index was recor-
cut into four parts and kept in the culture initiation media for another
ded in media with 33 µM BAP. The cultures appeared as
30 days. During the initial phase, cultures were subcultured three
times to reduce browning due to the presence of phenolic com- a clump of corms and produced small stunted shoot
pounds. The fresh weight and percentage of buds obtained from clusters (Figure 2). The results indicated that decreasing
each treatment were recorded. BAP to 22 µM significantly caused lower abnormality2448 Afr. J. Biotechnol.
Table 2. Shoot proliferation and elongation response to different combination of BAP with (+) and without (0) IAA in Musa
accuminata cv. Berangan.
Initiation media Proliferation media
Number of shoot Mean shoot length Multiplication rate
BAP (µM) BAP (µM) IAA (2 µM)
d f d
+ 14 ±1.4 7.73±0.6 3.5±0.3
33 33 d de d
0 12.75±3.7 6.43±0.9 3.18±0.9
f h f
+ 20.25±2.5 10.60±1 5.06±0.6
33 22 e g e
0 17.25±2.2 9.33 ±0.6 4.31 ±0.5
c e c
+ 7.75±1.2 6.52±0.7 1.93±0.3
33 11 c
0 7.25±2.6 5.8±0.7cde 1.81±0.6c
d gh d
+ 12.25±1.7 9.99±0.9 3.06±0.4
22 22 d g d
0 11±1.6 8.9±1 2.72±0.4
+ 7.5±2.5 c 5.26±1bcd 1.87±0.6c
22 11 bc bcd bc
0 6.75±1.5 5.7 ±0.5 1.68±0.3
+ 5 ±0.81abc 4.7±0.6abc 1.25±0.2abc
11 11 abc ab abc
0 5.25±0.95 4.32±0.8 1.31±0.2
ab
3.93±1.8a
ab
+ 4±0.81 1±0.2
11 6.5
0 3.5±1.2 a 3.98±0.2a 0.87±0.3a
Values are the mean ± SD. Values with different letters are significantly different at the 0.05 level.
f
f
e
cde
de
bcd bcd abc
ab
ab ab ab ab a
Figure 1. Effect of BAP pulsing with and without IAA on abnormality. Vertical bars indicate standard deviation (n = 4).
Values with different letters are significantly different at the 0.05 level.
index than 33 µM where normal shoot induction was the banana cultivar Brasilian (AAA). Previous researchers
highest. (Vuylsteke and De Langhe, 1985; Venkatachalam et al.,
This study had shown that increasing the concentration 2007; Bairu et al., 2008) indicated that 5 mg/l (22.2 µM)
of BAP during the initiation stage enhanced the fresh BAP was the optimum concentration for most banana
weight and percentage of buds formation. The importance cultivars. However, Arinaitwe et al. (2000) reported that in
of the application of high BAP concentration to initiate vitro bud initiation from banana was cultivar dependent.
bud formation from explants were reported by Zaffari et Upon subculture for shoot proliferation, the highest
al. (2000) and Subramaniam et al. (2008) in Cavendish number of normal and elongated shoots was derivedJafari et al. 2449
A B
C
Figure 2. A, B: Abnormality index caused by BAP. C: normal and elongated plantlet.
from media supplemented with 2 µM IAA and 22 µM high concentration of BAP after bud initiation was not
BAP. Generally, the results have shown that shoot multi- essential for shoot propagation due to the reduction in the
plication and elongation was significantly better in the number of shoots and high incidences of abnormality.
presence of IAA than BAP alone. Dhed’a et al. (1991) High concentrations of BAP did not allow recovery of the
reported that combinations of BAP with IAA or IBA were explants in tissue cultures in becoming complete normal
effective for in vitro multiplication of bananas and plan- plants due to the habituation effect of BAP. In addition,
tains. Resmi and Nair (2007) reported high shoot multipli- similar response was observed in terms of abnormality
cation but a reduction in the length of shoots in media index even with the incorporation of IAA to the medium
with a combination of BAP and IAA in triploid cultivar by (Figure 1). D’Amato (1978) reported that application of
using inflorescence explants. Venkatachalam et al. high concentrations of growth hormones for clonal propa-
(2007) reported a reduction in the number as well as gation is often disadvantageous since they may cause
length of shoot that occurred with exposure to high levels various chromosomal abnormalities resulting in the pro-
of BAP alone (44.44 µM) in banana cv. Nanjanagudu duction of non true-to-type plants. In this regard, there
Rasabale (AAB). Synergistic effects of plant growth regu- are numerous reports which show the shortcomings of
lators have influenced the cultural response in banana using high level of cytokinin that produces abnormality and
shoot proliferation and elongation. affects its genetic variability (Martin et al., 2006; Shirani
In this study, the abnormality index increased with et al., 2009).
increasing concentrations of BAP with the highest value It can be concluded that the application of high concen-
at 33 µM BAP (Figure 1). Vidya and Nair (2002) reported tration of BAP increased bud formation during the initiation
that occurrence of somaclonal variants in red banana stage. However, it might be deleterious to the cultures
(AAA) is due to the presence of high concentration of due to high abnormality index. Incorporation of IAA to the
BAP in the culture medium. This study suggested that media enhanced shoot proliferation and elongation but2450 Afr. J. Biotechnol.
could not reverse the abnormality index that has occur- Pua EC (2007). Banana In. Biotechnology in Agriculture and Forestry,
Vol. 60 Transgenic Crops V. Pua EC, Davey MR (Eds). Springer-
red. The application of BAP in the media needs to be Verlag. Berlin Heidelberg. pp. 3-34.
carefully monitored in establishing optimized culture sys- Resmi L, Nair AS (2007). Plantlet production from the male
tems for in vitro propagation to obtain normal plantlets. inflorescence tips of Musa acuminata cultivars from South India.
Plant Cell Tissue Organ Cult. 88: 333-338.
Rahman MZ, Sharoar MG, Matin MN, Rahman MH, Rahman MM, Islam
MR (2006). High frequency plant regeneration of a dessert banana
ACKNOWLEDGMENTS cv. mehersagar for commercial exploitation. Biotechnology, 5(3): 296-
300.
We would like to thank University Malaya for the financial Shirani S, Mahdavi F, Maziah M (2009). Morphological abnormality
among regenerated shootsof banana and plantain (Musa spp.) after
support by providing the grant and the author’s student
in vitro multiplication with TDZ and BAP from excised shoot tips. Afr.
fellowship. The authors would also like to thank Ms. Nur J. Biotechnol. 8(21): 5755-5761.
Ardiyana Rejab for her assistance in the preparation of Subramaniam S, Rathinam X, Poobathy R, Sinniah U (2008). In Vitro
this manuscript. Production of Multiple Bud Clumps (Mbcs) from Cavendish Banana
Cultivar, Brasilian (AAA). American-Eurasian J. Sustainable Agric.
2(3): 300-307.
Strosse H, Schoofs H, Panis B, Andre E, Reyniers K, Swennen R
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